Flow microsphere immunoassay for the quantitative and simultaneous detection of multiple soluble analytes.

Publisher Summary This chapter develops a quantitative and highly sensitive assay, named “flow microsphere immunoassay (FMIA),” permitting the simultaneous quantification of several analytes in a sample. The method employs several discrete size classes of plastic microspheres of 5, 7, 10, and 15 pm diameter, each class coated with a specific capture reagent. The four size classes of coated microspheres are mixed together and added to the sample under analysis. Each microsphere acts as an accumulator that removes from the sample and concentrates specific analyte for later measurement. Following incubation in the sample, the microspheres are washed to remove unbound sample and labeled with a fluorescent reagent that reacts only with the microsphere-accumulated analyte, labeling only those microsphere. Following washing, the microsphere suspension is analyzed using a flow cytometer that measures microsphere size and fluorescence. As it passes through the sensor region of a cytometer, the size of each microsphere is measured identifying the microsphere class and, thereby, the capture reagent. Fluorescence of the microsphere is measured; the amplitude of the signal indicates the quantity of the analyte in the sample.